Modified metal materials, surface modifications to improve cell interactions and antimicrobial properties, and methods for modifying metal surface properties

ABSTRACT

The present disclosure is directed to modified metal materials for implantation and/or bone replacement, and to methods for modifying surface properties of metal substrates for enhancing cellular adhesion (tissue integration) and providing antimicrobial properties. Some embodiments comprise surface coatings for metal implants, such as titanium-based materials, using (1) electrochemical processing and/or oxidation methods, and/or (2) laser processing, in order to enhance bone cell-materials interactions and achieve improved antimicrobial properties. One embodiment comprises the modification of a metal surface by growth of in situ nanotubes via anodization, followed by electrodeposition of silver on the nanotubes. Other embodiments include the use of LENS™ processing to coat a metal surface with calcium-based bioceramic composition layers. These surface treatment methods can be applied as a post-processing operation to metallic implants such as hip, knee and spinal devices as well as screws, pins and plates.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 12/246,455 filed Oct. 6, 2008, which claims priority to U.S.Provisional Patent Application No. 60/978,064 filed Oct. 5, 2007,entitled “SURFACE MODIFICATIONS TO IMPROVE BONE CELL-MATERIALSINTERACTIONS AND ANTIMICROBIAL PROPERTIES,” each of which isincorporated herein by reference.

The present application incorporates the subject matter of (1)International Publication No. WO/2007/124511, entitled “RESORBABLECERAMICS WITH CONTROLLED STRENGTH LOSS RATES,” filed Apr. 25, 2007; (2)U.S. Publication No. 2007/0203584 A1, entitled “BONE REPLACEMENTMATERIALS,” filed Feb. 14, 2007; and (3) U.S. Publication No.2009/0068272 A1, entitled “MESOPOROUS CALCIUM SILICATE COMPOSITIONS ANDMETHODS FOR SYNTHESIS OF MESOPOROUS CALCIUM SILICATE FOR CONTROLLEDRELEASE OF BIOACTIVE AGENTS,” filed Sep. 15, 2008, in their entiretiesby reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This work was partially funded by the Office of Naval Research (GrantNo: N00014-01-05-0583) and the National Science Foundation (Grant No:CTS-0134476), and the United States government has, therefore, certainrights to the present invention.

TECHNICAL FIELD

The present disclosure is generally directed to modified metal materialsfor implantation and to methods for modifying surface properties ofmetal substrates for enhancing cellular adhesion and promoting growth ofnatural bone cells while preventing microbial activities.

BACKGROUND

Bone and joint replacement materials have been useful for treating awide variety of musculoskeletal disorders. Replacement materials can,for example, be designed to restore both lost structure and function,particularly for load bearing applications. Bones in normal, healthycondition carry external joint and muscular loads by themselves.Following the insertion of orthopedic screws and/or implants, thenatural bone in the treated region will share its load-carrying capacitywith the implanted materials. Thus, the same load that had beenoriginally born by the bone itself will now be carried by the‘composite’ new structure. For load bearing screws and implants,clinically available devices are typically metallic.

The requirements for orthopedic metallic implants can be broadlycategorized as (1) biocompatibility between the material and thesurrounding environment with little or no adverse cytotoxicity andtissue reaction; and (2) the mechanical and physical propertiesnecessary to achieve the desired biophysical function. Some desiredproperties are, for example, low modulus, high strength, good ductility,excellent corrosion resistance in the body fluid medium, high fatiguestrength and good wear resistance. Titanium (Ti) and its alloys arewidely used in orthopedic and dental implants because of favorablemechanical properties, corrosion resistance, and biocompatibility.However, Ti is a bioinert material having minimal interaction with thesurrounding tissue. Accordingly, osseointegration with Ti implants,which requires a time-dependent kinetic modification of the surface ofthe implant, can take a long time.

Successful implantation challenges can also occur when the metallicimplant material is significantly stiffer than the adjacent bone.Internal load bearing functionality naturally performed by the bone, cannow be mainly supported by implanted screws or other structuralimplants. Such stress “shielding” of the natural bone can, in someinstances, alter the normal stress stimuli for bone growth, and thereduction of bone stresses relative to the natural situation causes boneto adapt itself by reducing its mass in a process of resorption aroundthe implant. This resorption/bone loss effect can cause micromotion ofthe screws/implants in response to external loads and could furtherdamage the interfacing bone layer and anchorage performances subsequentto possible loosening of the screw/implant [1].

Infection is also a possible side effect often associated with implantsand bone replacement surgeries. Infections, in some cases, may requireremoval of a surgically administered prosthesis or cause a significantdelay in post-surgical healing. This is often due to the accumulation ofmicrobial plaque or biofilm development on implants, screws or plates,which can contribute to recurrent infections as well as cause bone lossor prevent the necessary bone deposition for anchoring the surgicalimplant.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that advantages of the disclosure will be readily understood, adescription of aspects of the disclosure will be rendered by referenceto specific embodiments and the appended drawings. Understanding thatthese drawings depict only typical embodiments of the disclosure and arenot therefore to be considered to be limiting of its scope, thedisclosure will be described and explained with additional specificityand detail through the use of the accompanying drawings.

FIG. 1 is a FESEM image of nanoporous titanium oxide films anodized withan electrolyte-containing solution at 20V for 60 minutes in accordancewith an embodiment of the disclosure.

FIGS. 2A-C are, respectively, (A) a FESEM image of a silver coatednanoporous titanium oxide film surface, (B) a graphical representationof an EDS analysis of the elemental silver deposited on the Ti surfacemarked at (i) in FIG. 2A, and (C) a graphical representation of an EDSanalysis of a nanoporous surface region having minimal silver depositionmarked at (ii) in FIG. 2A in accordance with an embodiment of thedisclosure.

FIGS. 3A-B are, respectively, SEM micrographs of OPC1 cell morphology ona (A) nanoporous control surface, and (B) a silver-coated nanoporoussurface after 11 days of cell incubation in accordance with anembodiment of the disclosure.

FIG. 4 is a graphical representation of optical density measured at awavelength of 570 nm by a microplate reader following OPC1 cell cultureincubation with silver electroplated and non-electroplated nanoporous Tisamples for 11 days in accordance with an embodiment of the disclosure.

FIG. 5 is a schematic representation of a LENS™ processing device inaccordance with an embodiment of the disclosure.

FIGS. 6A-B are SEM micrographs of TCP coating layers on Ti substratesfabricated using LENS™ at a scan speed of 15 mm/sec. with a powder feedrate of 13 g/min. and at (A) 500 W laser power, and (B) 400 W laserpower in accordance with an embodiment of the disclosure.

FIGS. 7A-B are SEM micrographs of TCP coating layers on Ti substratesfabricated using LENS™ at 500 W power with a powder feed rate of 14g/min. and a scan speed of (A) 10 mm/sec., and (B) 15 mm/sec. inaccordance with an embodiment of the disclosure.

FIGS. 8A-B are SEM micrographs of TCP coating layers on Ti substratesfabricated using LENS™ at 500 W power at a scan speed of 10 mm/sec. andwith powder feed rate of (A) 9 g/min., and (B) 13 g/min. in accordancewith an embodiment of the disclosure.

FIGS. 9A-B are interfacial SEM micrographs of TCP coating layers on Tisubstrates fabricated using LENS™ at 500 W power with powder feed rateof 13 g/min. and a scan speed of (A) 10 mm/sec., and (B) 15 mm/sec. inaccordance with an embodiment of the disclosure.

FIGS. 10A-B are SEM micrographs of the microstructure of TCP coatinglayers on modified Ti substrates fabricated using LENS™ at 500 W power,10 mm/sec. scan speed and 9 g/min. powder feed rate and showing acomposite layer region (A) close to the metal substrate, and (B) alongan exterior surface of the coating layer in accordance with anembodiment of the disclosure.

FIG. 11 is a SEM micrograph of a Ti surface modified with a TCP coatinglayers using LENS™ at 400 W laser power, 15 mm/sec. scan speed and 13g/min. powder feed rate, and after polishing and etching in accordancewith an embodiment of the disclosure.

FIG. 12 is a graphical representation of hardness profiles of TCPcoating layers formed using LENS™ at 500 W laser power and a powder feedrate of 13 g/min. in accordance with an embodiment of the disclosure.

FIG. 13 is a graphical representation of X-ray diffraction spectra ofTCP coating layers using LENS™ and with a powder feed rate of 13 g/min.in accordance with an embodiment of the disclosure.

FIGS. 14A-D are SEM micrographs illustrating OPC1 morphology and celladhesion on Ti substrates after 5 days incubation in cell culture with(A) uncoated Ti, and (B) TCP coated Ti; and after 11 days incubation incell culture with (C) uncoated Ti, and (D) TCP coated Ti in accordancewith an embodiment of the disclosure.

FIG. 15 is a graphical representation of OPC1 cell proliferation onTCP-coated Ti substrates and uncoated Ti substrates in accordance withan embodiment of the disclosure.

DETAILED DESCRIPTION

The present disclosure describes embodiments and arrangements of metalsurface modifications for improving interactions between natural bonecells and the implanted materials, and for improving antimicrobial andantifungal properties for metal-based implants. In some embodiments,surface modification of titanium (Ti) materials can negatively influencecolonization of bacteria as well as promote interaction between humanosteoblast cells and the modified implantable material. In oneembodiment, Ti material surfaces can be oxidized to form a titaniumdioxide (i.e., TiO₂, titania) layer on the Ti substrate. For example,anodization of Ti substrate surfaces can result in in situ titaniaformation in a porous form, or in another embodiment, a nonporous form.In some embodiments, the titania layers and/or porous nanostructurespromote Human osteoblast (HOB) cell attachment and growth on themodified Ti substrate. In another embodiment, a laser engineered netshaping (LENS™) processing technique is described herein for coatingmetal implant surfaces (e.g., Ti surfaces) with uniformly distributedcalcium phosphate-based bioceramics (or other calcium-based bioceramics)under optimized parameters to achieve a greater coating thickness. In afurther embodiment, antimicrobial and/or antifungal particles can bedeposited on the surfaces of the Ti material, a TiO₂ layer (e.g., aporous nanotube structure layer, a nonporous film, etc.), acalcium-based bioceramic coating layer, etc., to promote antimicrobialproperties such as resistance to growth of Gram negative bacteria (e.g.,Pseudomonas aeruginosa). In some embodiments, use of these surfacetreatment methods can be used to create coatings with compositiongradients across the coating thickness, which can significantly reduceinterfacial problems associated with a sharp interface that is typicallypresent in conventional coating processes.

It will be appreciated that several of the details set forth below areprovided to describe the following embodiments in a manner sufficient toenable a person skilled in the relevant art to make and use thedisclosed embodiments. Several of the details and advantages describedbelow, however, may not be necessary to practice certain embodiments ofthe disclosure. Additionally, the disclosure can include otherembodiments that are within the scope of the claims but are notdescribed in detail with respect to FIGS. 1-15.

A. Embodiments of Modified Metal Implant Materials and Methods forPreparing and Using Such Materials

In one aspect, the present disclosure is directed to methods forproducing bone replacement materials with improvedantimicrobial/antifungal properties and enhanced cellular adhesion(tissue integration), and to bone replacement materials producedtherefrom. Surface modifications to enhance the interactions betweenbone cells and implant materials, e.g., Ti-based materials, aredescribed herein. Some embodiments comprise surface coatings for Timetal implants fabricated using both (1) electrochemical processingand/or anodization, and (2) laser processing, in order to enhance bonecell-materials interactions and achieve improved antimicrobialproperties. In some embodiments, the surface treatment methods describedherein can be applied as a post-processing operation to metallicimplants such as hip, knee and spinal devices as well as screws, pinsand plates.

Specific embodiments of suitable implant material compositions caninclude, but are not limited to; metals (e.g., titanium (commerciallypure Ti, and both a and 13 alloys); aluminum (Al), iron (Fe), vanadium(V), etc.); metal alloys (e.g., Ti alloys with major alloying elementssuch as Al, V, Nb, Fe, Zr, Mo, O, Ni, Cr, Co; Ta forming alloys such asTi6A14V, Ti-6Al-7Nb, Ti-5Al-2.5Fe, Ti-12Mo-6Zr-2Fe, Ti-15Mo-5Zr-3Al,Ti-15Mo-3Nb-3O, Ti-13Nb-13Zr, Ti-35Nb-5Ta-7Zr; Stainless steel, CoCrMO,etc.), metal oxides (e.g., TiO₂); ceramics, inorganic salts (e.g., suchas different forms of calcium phosphates and calcium carbonates andtheir combinations); polymeric materials and/or combinations thereof.

In one embodiment, modification of a metal implant surface (e.g., a Tisurface) includes fabrication of a TiO₂ surface layer for enhancingtissue integration. In some embodiments, the TiO₂ layer can be a porouslayer having in situ titania nanotubes formed on a surface of a metalsubstrate. In other embodiments, the TiO₂ layer can be a nonporous filmdeposited on the metal substrate [16]. In one example, a TiO₂ layerhaving a thickness of approximately 50 nm or greater can positivelyinfluence natural bone cell behavior (attachment, spreading,proliferation, survival, etc.). TiO₂ can be deposited on a Ti surfaceusing a variety of methods, such as thermal oxidation andelectrochemical oxidation (e.g., anodization). In some arrangements, theprimary composition of the porous or nonporous layer resulting fromthese methods includes TiO₂. In some embodiments, however, thecomposition can include small amounts of ions such as Na⁺, K⁺, Mg²⁺,Zn²⁺, Sr²⁺, Si²⁺, Ca²⁺, or other metal ions, in a range of approximately0-10 mol % of TiO₂.

In other embodiments, laser engineered net shaping (LENS™), which is acommercial, rapid prototyping (RP) process, can be used to coat a metalimplant surface (e.g., a Ti surface) with uniformly distributed calciumphosphate (CaP). In one embodiment, the coating layer can include atleast 60% of mixed phases of calcium phosphates, such as tricalciumphosphate (TCP), tetra-calcium phosphate and hydroxyapatite. In someembodiments, the remainder of the coating layer can include acombination of base metal calcium phosphate compounds such as titaniumphosphate, titanium oxide and calcium-titanium phosphate. In otherembodiments the coating layer can include other resorbable ceramics,such as ceramics containing calcium phosphates (CaP), calcium sulfates(CaS) and calcium silicate (CaSi) compositions, alone or in combination,to improve the interactions between bone cells and implant materials. Inanother embodiment, phases of calcium phosphates incorporated in thecoating layer can be in a crystalline form, an amorphous form, or acombination of crystalline and amorphous forms. Further, the compositionof the coating layer can have a continuous concentration gradient from ametal substrate—coating interface to a coating surface (e.g., a lowpercentage of calcium-based bioceramic particles at the interface to ahigh percentage of calcium-based bioceramic particles at a coatingsurface).

TCP (Ca₃(PO₄)₂) is a CaP-based synthetic material that can form abioactive bond with natural bone. Compared with hydroxylapatite, TCP hasa lower calcium-to-phosphorous ratio, which can increase the degradationrate when the ceramic is placed in a biological environment, such asbody fluid. TCP degrades in the body and the products are subsequentlyresorbed by the surrounding tissue. Therefore, such matrix absorptionmay be used to expose underlying surfaces to natural bone cells (e.g.,osteoblast cells) or to release admixed materials such as antibiotics,growth factors (e.g., human growth factors, osteoinductive growthfactors), or other biological agents (e.g., proteins, morphogens,pharmaceutical drugs, vitamins, etc.) in controlled drug release.

In one embodiment, compositions including CaP and CaSi can be useful forproviding compositions having selectable controlled release profiles forbioactive agents, selectable controlled strength loss rates within aselect period of time, etc. For example, mesoporous CaSi particles, suchas those described in U.S. Publication No. 2009/0068272, and which isincorporated in its entirety by reference, can be included in thecoating layer for controlled release of bioactive agents. In someembodiments, a calcium-based composition for fabricating a bioceramiccoating layer using LENS™ can include a Ca-based ceramic (e.g., CaP,CaS, CaSi) having at least one dopant included therein for providingselectable controlled strength loss rates. Examples of suitable dopantscan include metal salts with metal ions (e.g., Zn²⁺, Mg²⁺, Si²⁺, Na⁺,Sr²⁺, Cu²⁺, Fe³⁺/Fe²⁺, Ag⁺, Ti⁴⁺, CO₃ ²⁻, F⁻), and in anotherembodiment, the dopant can include metal oxides (e.g., MgO, ZnO, NaF,KF, FeO/Fe₂O₃, SrO, CuO, SiO₂, TiO₂, Ag₂O and CaCO₃).

In accordance with one embodiment of the disclosure, calcium-basedbioceramic coatings (e.g., TCP, tetra-calcium phosphate, hydroxyapatite,CaSi, CaS, etc.) can be prepared on a commercially pure Ti (cp-Ti)substrate using a LENS™ system and technique. The LENS™ process can beused to coat the surface areas of metal implant materials with a highdegree of accuracy, and desired physical and mechanical properties ofthe implant coating are attainable through appropriate selection ofLENS™ process parameters (e.g., laser power, laser scan speed,bioceramic powder feed rate, etc.). In another embodiment, thebioceramic coating technique using LENS™ can also be used to repair adamaged coating due to its selectivity and precision. In somearrangements, a bioceramic composite coating layer can have a coatinglayer thickness of approximately 200 gm to approximately 700 In otherarrangements, the composite coating layer thickness can be between about50 μm and about 900 μm, or from about 200 μm to about 400 μm. In furtherarrangements, the composite coating layer thickness can vary across ametal surface.

During the LENS™ coating process, a Nd:YAG laser can melt a surface of aTi substrate (or other metal substrate). Simultaneously, calciumphosphate powder (or other calcium-based biocompatible ceramic powder)can be fed or delivered to the melted surface to mix with the moltenmetal and create a composite layer (e.g., a CaP-Ti composite layer). Aslaser power and/or powder feed rate increases, the thickness of thecomposite bioceramic coating can increase. In one embodiment, the volumeof molten metal created due to laser heating can be a combined effect oflaser power and interaction time between the laser and the material. Forexample, an increase in laser power (while other parameters are heldconstant) can transfer more heat energy to the substrate, which canincrease the liquid-metal volume yielding a greater composite layerthickness. Similarly, an increase in laser scan speed can reduce theinteraction time between the laser and the substrate thereby decreasingthe amount of molten metal. Accordingly, the composite layer coatingthickness can be increased or decreased as a function of laser scanspeed.

In another embodiment, varying the powder feed rate while using LENS™with a constant laser power and constant scan speed can yield varyingcomposite bioceramic coating thicknesses even though the surface volumeof liquid metal generated by the laser is substantially constant. Forexample, a decrease in powder feed rate can yield a significant decreasein composite coating thickness (e.g., about 100 μm). Similarly, a higherpowder feed rate can be used to deliver more calcium-based bioceramicpowder (e.g., TCP powder, HAp primary phase CaP powder, etc.) directlyinto the molten metal pool, increasing both the concentration ofcalcium-based bioceramic in the coating as well as increasing the totalcomposite bioceramic coating thickness.

In a further embodiment, the volume fraction of calcium-based bioceramicin the coating can be strongly influenced by scan speed. Operation ofthe LENS™ laser at a slower scan speed can deliver more calcium-basedbioceramic powder to the molten metal pool than operation at a fasterscan speed. Moreover, higher interaction time (between laser and metal)at a slow scan speed can increase the volume of the molten metal pool onthe substrate, which can then accommodate a proportionately greateramount of powder. Accordingly, calcium-based bioceramic (e.g., TCP,tetra-calcium phosphate, hydroxyapatite, CaSi, CaS, etc.) loading in thecoating can increase with decreasing laser scan speeds.

In one embodiment, the coating layer includes one or moremicrostructures formed by the solidification of molten metal andbioceramic particles. In some embodiments, microstructure formation canvary within the composite coating layer created using the LENS™ process.For example, in coating regions closer to the composite coating-metalinterface, the grain structures can have a columnar orientation. As thegrain structures move along the thickness of the coating layer from theinterface region to the exterior of the coating layer, the grainstructures can transition to an equiaxed grain structure. Additionally,when a laser scan speed is reduced, a composite coating hardness valuecan correspondingly increase due to an increase in the volume fractionof calcium-based bioceramic in the coating.

In accordance with the present disclosure, embodiments of bioceramiccomposite coatings can provide improved biocompatibility between metalimplants (e.g., Ti metal implants) and human osteoblast cells (HOB), byenabling cell attachment and proliferation. Further, embodiments ofbioceramic composite coatings may also promote cell differentiation,extracellular matrix (ECM) formation and biomineralization.

In a further embodiment, antimicrobial and/or antifungal agents (e.g.,silver, zinc oxide, aluminum oxide, copper oxide, and their combination,etc.) can be deposited on the surfaces of a metal implantable material,or a modified metal material having a TiO₂ layer (e.g., a porousnanotube structure layer, a nonporous film, etc.) and/or a calcium-basedbioceramic coating layer to promote antimicrobial and antifungalproperties. For example, a modified or non-modified Ti surface having asilver electroplated coat can effectively inhibit greater than 99% ofPseudomonas aeruginosa colony growth. Non-modified or modified Tisubstrate surfaces without silver deposition do not demonstrate theseinhibitory properties against colony formation and growth of P.aeruginosa.

In one embodiment, electrodeposition techniques can be used to depositsilver (Ag) on a titania nanotube surface, a TiO₂ nonporous film layer,a bioceramic composition coating, or other surface of a metal substrateto inhibit bacterial colony growth and/or fungal overgrowth. In oneembodiment, a TiO₂ surface layer or a bioceramic composition coating,with or without silver deposits, can facilitate cellular attachment,high cell proliferation rates and enhanced bone cell-materialinteractions when compared to a non-modified Ti surface. Antimicrobialand/or antifungal agents can be deposited such that the deposits do notform a continuous coating on the metal surface, TiO₂ surface,calcium-based bioceramic composition coating, or other surface. Forexample, the antimicrobial and/or antifungal agents can be deposited, byany known deposition technique, in interconnected or particulate formswith particle sizes in the range of about 1 nm to about 100 microns.

In another embodiment, a metal device for attaching to bone includes acomposite metal structure configured to be implanted in the body. Themetal device can include a first surface region of the metal structurehaving titania nanotubes. The metal device can also include a calciumphosphate-based bioceramic surface coating on a second surface region ofthe composite metal structure. In a further embodiment, the first and/orsecond regions can have an antimicrobial and/or antifungal agentdeposited thereon. In one arrangement, the first and second surfaceregions are separate regions. In another arrangement, the first surfaceregion and the second surface region are at least partially overlapping.Another aspect of the disclosure is directed to a method for modifying asurface of a metal device for implanting in a body. The method caninclude oxidizing a metal surface of the device to form a TiO₂ layer,such as a porous layer having nanotube microstructures, or a non-porouslayer having a titania film, on at least a portion of the surface.Following oxidation, the method can also include electrodepositing anantimicrobial and/or antifungal agent (e.g., silver) onto the metalsurface of the device. Optionally, or in lieu of the oxidizing step, themethod can include using a laser processing technique (e.g., LENS™) tocoat one or more surface regions of the device with a calcium-basedbioceramic surface coating. If desired, an antimicrobial and/orantifungal agent may be deposited onto the calcium-based bioceramicsurface coating.

In a particular example, a hip joint implant can have a stem portion forinserting into a femur bone, a ball portion for replacing the headportion of the natural femur bone and a cup portion for replacing thepatient's hip socket. The stem portion of the implant can be made ofmetal, such as titanium, having surface modifications for improving bonecell-materials interactions and/or inhibiting bacterial growth fordiminishing risks related to post-surgical infection. In one embodiment,the stem portion could have single surface modification (e.g., titaniananotubes, silver deposition, bioceramic coating), or in otherembodiments, the stem portion may include a combination of titaniananotube microstructure, silver deposition, bioceramic coating and/orother surface modification. Moreover, because the accuracy of themethods described herein, surface modifications and combinations ofsurface modifications can be in a complex pattern. Furthermore, thesurface modifications described herein can be combined with other knownmodification used in the relevant art. In the example of a hip implant,the ball and socket portions may or may not include metal and may or maynot include surface modifications. One of ordinary skill in the art willrecognize other forms of implants that can have surface modifications,other body regions for attaching and/or inserting metal implants, aswell as other attachment mechanisms (e.g., screws, pins, plates, etc.).

One of ordinary skill in the art will recognize that methods disclosedherein can be used to modify implantable metal devices having any sizeand shape. Furthermore, some embodiments of methods for modifying metalsurfaces described herein may include modifying only a portion of thesurface. Furthermore, implantable metal devices may include a firstmodified surface having a first modification and a second modifiedportion having a second modification. While sample sizes, shapes, andforms of implantable metal are disclosed herein in the context ofexamples, one of ordinary skill in the art will recognize other sizes,shapes and forms of samples that can be used as examples and/or asimplantable metal devices.

B. EMBODIMENTS AND EXAMPLES OF METHODS FOR ELECTROCHEMICALLY PROCESSINGSURFACE COATINGS ON TI IMPLANTS, AND CHARACTERIZATION OF TI METALSUBSTRATES HAVING SUCH SURFACE MODIFICATIONS

The following examples are intended to demonstrate aspects of thedisclosure more fully without acting as a limitation upon the scope ofthe disclosure, as numerous modifications and variations will beapparent to those skilled in the relevant art.

In one embodiment, the surfaces of metal implantable materials, forexample Ti metal materials, can be modified to enhance interactionsbetween bone cells and the metal implantable materials as well as toincrease antimicrobial properties for improving post-surgical healingand protecting the patient from postoperative infection.

Preparation of a Titania (TiO₂) Layer on Ti Surface Example 1

Commercially pure titanium (cp-Ti, 99.6% pure) sheets of 0.5 mmthickness were used as a starting material. In this example, circular Tidiscs were cut having a 12 mm diameter and the samples were abraded withsilicon carbide paper in successive grades from 600 to 1200 grit (LecoCorporation, MI) followed by ultrasonic cleaning in distilled water andair drying at room temperature. The Ti discs were then polished with acotton polishing cloth using a 1 μm alumina suspension. Following thepolishing step, the samples were anodized in a two-electrodeelectrochemical anodization cell, with a titanium anode and platinumcathode, and in an electrolytic aqueous solution containingapproximately 0.1 mole/L sodium fluoride and approximately 1.0 mole/L ofsulfuric acid at a constant dc voltage of 20V for about 60 minutes.During anodization, the electrolytic solution was stirred with amagnetic stir bar. Surface and lateral topography of the modified Tisamples was visualized using a field-emission scanning electronmicroscope (FESEM; FEI, Sirion, OR) fitted with an energy dispersivespectroscopy (EDS) detector.

Anodization of Ti in the above-defined electrolytic aqueous solutioncontaining sodium fluoride and sulfuric acid can result in a nanoporousmorphology. FIG. 1 is a FESEM image of a cross-sectional view ofnanoporous titanium oxide films anodized with the electrolyte-containingsolution at 20V for 60 minutes in accordance with an embodiment of thedisclosure. As shown in FIG. 1, examination of the cross-sectional viewof the Ti substrate revealed the presence of nanotube structures. Theaverage internal diameter of the nanotubes was 100 nm and the averagelength was 300 nm.

Nanotubes can be formed by two simultaneous processes: (1) anelectrochemical etch, and (2) chemical dissolution [7, 8]. Duringelectrochemical etching, an initial oxide layer forms on a Ti surfacedue to the interaction between Ti⁴⁺ and O²⁻ ions. In the presence of Fions, oxide layers dissolve partially and nanometer sized pits areformed. At the base of the pits, both chemical dissolution andelectrochemical etching can take place to form a thin barrier layer,which can increase the electric-field intensity resulting in furtherpore growth. As the barrier layer decreases, and higher electric fieldintensity causes further electrochemical oxidation and dissolution,separate channels are formed and give rise to the final nanotubestructure shown in FIG. 1.

The specific electrolyte composition chemistry described in Example 1was chosen following experimentation using a plurality of electrolytecompositions [9]. As a result of such experimentation, the inventorsfound that two specific electrolyte criteria are desirable for formingnanoporous morphology: (1) the presence of fluoride ions, and (2) acidicsolutions. Sodium fluoride (NaF) completely dissociates in wateryielding F⁻ ions to form HF and OH⁻ ions. HF is a weak acid in an acidicsolution with a dissociation constant (K_(a1)) of 6.8*10⁻⁴ which is lowin comparison to strong acids like H₂SO₄ which has a second dissociationconstant (K_(a2)) of 1.2*10⁻², or H₃PO₄ which has a third dissociationconstant (K_(a3)) of 7.5*10⁻³. Because most of the F⁻ ions exist in theform of HF, the presence of strong acids in the electrolytic solutionprevents HF from dissociation, thus inhibiting high chemical dissolutionand thereby allowing the nanotube array structure to be maintained. Oneof ordinary skill in the art will recognize other electrolyte solutionsfor forming nanotube microstructures as well as additional anodizationconditions and arrangements.

Electrodeposition of Silver

In another embodiment, silver (Ag) can be electrically deposited ontothe surface of Ti.

Example 2

In a specific example described herein for purposes of illustration andin accordance with an embodiment of the disclosure, six anodized sampleswere cleaned and anodized a second time with approximately 0.01 M silvernitrate solutions at 5V for about 2 minutes to produce silver depositedTi samples. In this example, the silver deposited samples are denoted asAg-1, Ag-2, Ag-3, Ag-4, Ag-5 and Ag-6. Anodized Ti samples withoutsilver deposition (e.g., control samples) are referenced as C-1, C-2,C-3, C-4, C-5 and C-6. FIGS. 2A-C are, respectively, (A) a FESEM imageof a silver coated nanoporous titanium oxide film surface, (B) agraphical representation of an EDS analysis of the elemental silverdeposited on the Ti surface marked at (i) in FIG. 2A, and (C) agraphical representation of an EDS analysis of a nanoporous surfaceregion having minimal silver deposition marked at (ii) in FIG. 2A inaccordance with an embodiment of the disclosure. As shown in FIG. 2B,the samples anodized with the silver nitrate solution demonstrate thepresence of elemental silver on the nanoporous titania surface. As shownin FIG. 2C, in regions of the nanotube titania surface not covered byelemental silver deposits, elemental Ti and O are visible.

Bone Cell-Materials Interactions

Interactions between natural bone cells and the metal implant materialscan be characterized with in vitro biocompatibility assessments using ahuman osteoblast (HOB) cell line. HOB cells can be derived from anosteoblastic precursor cell line (OPC1) established from human fetalbone tissue.

Example 3

In this example, cells were plated at a density of approximately 10⁵/cm²in 100 mm tissue culture plates and cultured in McCoy's 5A medium (withL-glutamine, without phenol red and sodium bicarbonate) and supplementedwith 5% fetal calf serum (FCS), 5% bovine calf serum (BCS), 2.2 gm/litersodium carbonate, 100 mg/liter streptomycin, and 8 μg/ml Fungizone(Gibco™ Laboratories, Grand Island, N.Y.). Cells were maintained at 37°C. under an atmosphere of 5% CO₂ and 95% air.

To examine the interactions between bone cells (e.g., OPC1 cells) and ananoporous titania surface, anodized nanoporous TiO₂ samples with andwithout a silver coating, as described in Examples 1 and 2 above, wereautoclaved at about 121° C. for 45 minutes. Following the autoclavingstep, OPC1 cells were seeded from the cultured plate to a top surface ofthe autoclaved samples in new culture plates. For example, OPC1 cellswere cultured on a series of control nanoporous surfaces (C-seriesdescribed above) and on a series of silver-coated nanoporous surfaces(Ag-series described above). The cell-seeded samples were maintained at37° C. under an atmosphere of 5% CO₂ and 95% air. Culture media waschanged every two days for all culture sample plates. In the exampledescribed herein, all OPC1 cells originated from the same cell linepassage and all plates were kept under identical conditions.

The OPC1 cells were cultured with the C-series and Ag-series samples foreither 5 or 11 days before the samples were fixed. The samples werefixed with 2% paraformaldehyde/2% glutaraldehyde in 0.1 M cacodylateovernight at 4° C. Following a rinse in 0.1 M PBS, each sample was fixedin 2% osmium tetroxide (OsO₄) for two hours at room temperature.Following the fixation steps, the samples were rinsed with 0.1Mcacodylate and dehydrated using an ethanol (EtOH) series for 10 minuteseach. Bone cell-materials interactions were evaluated for each of thefixed samples using scanning electron microscopy (SEM Hitachi's 570).

FIGS. 3A-B are, respectively, SEM micrographs of OPC1 cell morphology ona (A) nanoporous control surface, and (B) a silver-coated nanoporoussurface after 11 days of cell incubation in accordance with anembodiment of the disclosure. In both samples, the OPC1 cellsdemonstrate a filamentous network structure with cell-cell attachmentand cell spreading along the nanoporous surface. As shown in FIG. 3A,nodule formation was pronounced on nanoporous surfaces indicating earlycell differentiation during 11 days in cell culture. As shown in FIG.3B, cell morphology on silver-coated samples (Ag-series) demonstratesthat the number of interactions between OPC1 cells and thesurface-treated material were diminished when compared to the nanoporoussurface (C-series) without a silver coating. As demonstrated in thisexample, silver deposition decreased the amount of cell-materialinteraction; however, this observation is not attributed to cell death.In this example, no cell death was documented for the samples (C-seriesor Ag-series) incubated over 5 days or 11 days. Therefore, no adversetoxicity effects were observed due to the Ag-coating on the samplesurfaces.

Cell Proliferation

Cell proliferation can also be considered in a determination of metalimplant material in vitro biocompatibility.

Example 4

In this example, OPC1 cell proliferation on either Ti control samples (3samples each of Ag-coated [Ag—Ti] and non-coated [Ti]) or nanoporous Tisurfaces (3 samples each from the C-series and Ag-series) were evaluatedusing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) assay. MTT (Sigma, St. Louis, Mo.) solution at 5 mg/ml wasprepared by dissolving MTT in PBS followed by filter sterilization. TheMTT solution was then diluted by transferring 50 μl of the sterileconcentrated solution into 450 μl of serum free phenol red-freeDulbeco's minimum essential (DME) medium, which was then transferredinto each of the test wells (each containing an anodized Ti sample) of a12-well tissue culture plate for the cell proliferation assay. Duringthe MTT assay, cellular metabolic activity can convert the tetrazoliumin the MTT solution to formazan products. In this example, formazanformation was allowed to proceed for 2 hours at 37° C. The level ofcellular metabolic activity was determined by extracting the formazanproducts in 500 μl of a solubilization solution (10% triton X-100, 0.1NHCl and isopropanol). Following extraction, 100 μl of the solubilizationsolution was transferred to a fresh 96 well plate and the opticaldensity of the solution in each well was measured at a wavelength of 570nm using a Microplate reader (Cambridge Tech. Inc., Model 700 EIA). Thedata was presented as a mean optical density value with a standarddeviation for each sample.

FIG. 4 is a graphical representation of the optical density of theextracted formazan products measured at a wavelength of 570 nm by amicroplate reader following OPC1 cell culture incubation with theanodized and non-anodized Ti samples with and without silverelectroplated surfaces for 11 days in accordance with an embodiment ofthe disclosure. As shown in FIG. 4, silver electrodeposited samples,Ag-series and Ag—Ti, showed lower cell density, and therefore reducedcellular proliferation, when compared to the samples without silverelectroplating, C-series and Ti, respectively. Moreover, nanoporoussurfaces (Anodized) showed higher cell density, and therefore increasedcellular proliferation, in comparison to the non-nanoporous (Control)samples for both silver deposited and non-silver coated samples.

Antimicrobial Tests

As noted above, bacterial infection and/or fungal overgrowth can be aside effect often associated with implants and bone replacementsurgeries.

Example 5

In this example, bacterial growth tests using the Gram-negativebacteria, Pseudomonas aeruginosa (ATCC 9027) was used to assessantimicrobial properties associated with the modified metallic implantsubstrates of Examples 1 and 2 described above. The bacterial growthtests described in this example were conducted using P. aeruginosabecause approximately 80% of infections associated with metallicimplants are caused by Gram-negative bacteria.

In this example, 12 mm discs of silver-coated anodized Ti substrates(Ag-series) and anodized Ti substrates without silver (C-series) wereplaced into individual wells of sterile tissue culture plates. Thesamples were then challenged with a volume to surface area ratio of 0.6ml bacterial inoculum/cm². For example, to each well containing the Tisamples, 1.2 ml of inoculum prepared in M101 medium (see Table 1) andcontaining ˜1×10⁵ colony forming units (cfu) of P. aeruginosa wereadded. The inoculum was prepared by diluting an overnight broth cultureof P. aeruginosa in M101 medium to yield an initial inoculum of ˜1×10⁵cfu/ml. The multi-well plate was then covered with a sterile lid andincubated at 37° C. for 24 hours. Following incubation, the supernatantfluid from each well was appropriately diluted and plated on Trypticasesoy agar (TSA) plates. The plates were then incubated at 37° C. for 24hours and surviving colony-forming bacteria were counted. The logreduction resulting from modified Ti substrates was calculated bysubtracting the log survivors of the test wells from the log survivorsof control wells (e.g., unmodified Ti substrates).

TABLE 1 Components of the M101 Challenge Medium Grams Component A Gramsper liter Component B per Liter Sodium citrate 1.3 Calcium Chloride 1.3Potassium phosphate 1.3 Magnesium chloride 1.3 Sodium oxalate 2.0 Sodiumchloride 9.8 Urea 25.0 Sodium sulfate 4.6 Potassium Chloride 3.2Ammonium chloride 2.0 Urea 25.0 Sterilize each solution and then combineat equal parts for use.

Table 2 shows results of antimicrobial activity for three bacterialdilution levels following a 24 hour incubation period. Referring toTable 2, the C-series samples demonstrated no antimicrobial effects asevidenced by the high number of surviving bacterial colonies in eachC-series well. In contrast, each well containing an Ag-coated 12 mmdiameter disk sample (e.g., Ag-1 to Ag-6) demonstrated superiorantimicrobial properties when compared to the non-coated samples Forexample, only one Ag-coated sample (Ag-1) had surviving colonies (e.g.,30 colonies; average: 30/3=10 colonies) while the assay plating resultsfrom the remaining Ag-coated samples were devoid of colonies. Therefore,log reduction in bacteria colony forming units (cfu) due to the silvercoating on modified Ti disk substrates can be estimated as the logarithmof the ratio of initial bacterial cfu to the average number of finalsurviving colonies. The resultant value in this example was 4.94 (i.e.,log ratio reduction>4 equates to approximately a>99.99% reduction inbacteria), demonstrating a very strong antimicrobial efficacy for thesilver-coated substrates. In contrast, control nanotube Ti samples(C-series) demonstrated no antimicrobial properties compared tobacterial growth in medium alone as indicated by a comparable survivingbacterial count at the 1×10⁻⁵ dilution level in the C-series samples andin the medium alone.

TABLE 2 Results of the antibacterial capacity of Ag electrodepositedsamples against the growth of bacterial colonies after 24 hours 24 hcount Test 0 time count Dilution Sample cfu/ml 1 × 10⁻¹ Dilution 1 ×10⁻³ Dilution 1 × 10⁻⁵ Ag-1 8.9 × 10⁵ 3 0 0 Ag-2 8.9 × 10⁵ 0 0 0 Ag-38.9 × 10⁵ 0 0 0 Ag-4 8.9 × 10⁵ 0 0 0 Ag-5 8.9 × 10⁵ 0 0 0 Ag-6 8.9 × 10⁵0 0 0 C-1 8.9 × 10⁵  TMTC* TMTC 1064 C-2 8.9 × 10⁵ TMTC TMTC 976 C-3 8.9× 10⁵ TMTC TMTC 1248 C-4 8.9 × 10⁵ TMTC TMTC 1016 C-5 8.9 × 10⁵ TMTCTMTC 1288 C-6 8.9 × 10⁵ TMTC TMTC 1216 Medium 8.9 × 10⁵ TMTC TMTC 1236Medium 8.9 × 10⁵ TMTC TMTC 1128 *The zero time plate count was 8.9 × 10⁸cfu/mL, TMTC = Too Many To Count

Two possible explanations for the observed antimicrobial properties ofAg can be that (1) the silver metal can react with water and releasesilver ions which may then combine with sulphydryl groups of respiratoryenzymes or nucleic acids in the bacteria, resulting in a respiratoryblock and ultimately causing death of the bacterium [10], and (2) silvermay react with the oxygen dissolved in the medium and generate activatedoxygen O* which may decompose the bacterium [11-14].

The results demonstrated in the above Examples show that Ti substratesurfaces can be modified by formation of titania nanotubes to enhanceinteractions between bone cell and implantable metal materials. Further,the nanoporous surface can be silver-coated to enhance antimicrobialproperties on the surfaces of the implantable materials. Accordingly,the results from the examples described above confirm that surfacemodification can improve both osseointegration and reduce chances ofinfection, which are advantageous properties for metallic devices thatcan be used for a variety of biomedical applications including metalimplants.

C. EMBODIMENTS AND EXAMPLES OF METHODS FOR LASER PROCESSING OF SURFACECOATINGS ON TI IMPLANTS, AND CHARACTERIZATION OF TI METAL SUBSTRATESHAVING SUCH SURFACE MODIFICATIONS Overview of a LENS™ Processing forImproving Biocompatibility

Compounds having bioresorbable properties encourage bone growth andfacilitate integration with bone tissue. Both in vitro and in vivostudies have shown that bioactive calcium phosphate-based ceramics arebiocompatible and osteoconductive; however, these ceramic materials arebrittle in nature and can only be used as a coating or as bone fillers.The usefulness of a coated implant depends on the stability of thecoating which is governed by its physical and mechanical properties. Oneaspect that is useful to address for any coated implant is the long termadherence of the coating with the substrate. A coating which separatesfrom the implant in vivo would provide no advantage over an uncoatedimplant and the resultant debris material from the coat separation canrender these coating systems even less desirable for use within thebody. A variety of different techniques have been used to coat metallicimplants with calcium phosphate-based ceramics such as, for example, dipcoating, sol-gel, electrophoretic deposition, biomimetic coating,simultaneous vapor deposition, pulsed laser deposition and plasmaspraying. The success of these coating processes can depend on theability to achieve high crystallinity within the coatings, goodadherence between the ceramic and the metal, control over coatingthickness and the ability to coat porous and complex shapes. Among them,an electrophoretic deposition process has been most widely used to coatporous and complex shaped implants. However, high temperature sinteringof such electrophoretically deposited coatings can often lead tocracking at the substrate-coating interface.

Dip coating and sol-gel processes are good for getting a thin coating onimplants, but achieving a thicker coating is often very difficult.Plasma spray calcium phosphate-based ceramic coatings suffer from lowcrystallinity and poor interfacial bonding. In addition, a high coolingrate can introduce cracks in the coatings which can reduce the adhesionstrength between the substrate and the coated ceramic.

Bioactive calcium phosphate-based ceramics, especially hydroxyapatite(HAp) and tricalcium phosphate (TCP), have chemical and crystallographicsimilarity to natural bone. TCP may have applicability in bonereconstruction and remodeling due to its bioresorbable properties, readyavailability and controlled size variation. Among these applications,coating on metallic implants can improve tissue integration of thecoated implants by providing a bioactive surface on otherwise bioinertmaterial, which can improve healing time. Examples of such coatings canbe found in International Publication No. WO/2007/124511, U.S.Publication No. 2007/0203584 and U.S. Publication No. 2009/0068272, eachof which is incorporated in their entirety by reference.

In accordance with one embodiment of the disclosure described herein,TCP coatings can be prepared on a commercially pure Ti (cp-Ti) substrateusing LENS™, a laser engineered net shaping and rapid prototyping (RP)process. TCP can be used as a representative material from the calciumphosphate family of bioceramics for which 45-150 micron size powders,desirable for LENS™ processing, can be readily available. However, thisprocess can be easily extended to other calcium phosphate, calciumsilicate and calcium sulfate-based materials.

Laser beams, owing to their high coherence and directionality, have theability to locally melt the surface of a metal substrate. A schematicrepresentation of the LENS™ processing device 100 is shown in FIG. 5.The device can include a Nd:YAG (neodymium-doped yttrium aluminumgarnet) laser that can have laser power 102 focused onto a metalsubstrate 104 to create a molten metal pool on the substrate surface106. The laser beam 102 can be focused on the substrate surface 106 bymoving one or more focusing lenses 108 along a Z-positioning axis 110.Powder (e.g., calcium phosphate powder) can then be injected into themetal pool from one or more powder delivery nozzles 112 associated withthe LENS™ processing device 100. As the molten metal cools, thecomposite bioceramic powder-metal composite later subsequentlysolidifies. The substrate 104 can then be scanned relative to thedeposition head to a write line of the metal having a finite width andthickness. Back and forth rastering (e.g., using an X-Y positioningstage 114) can create a pattern with fill material (e.g., powder) in adesired area to allow a layer of material to be deposited. Thisprocedure can be repeated several times until an entire solid ortailored porosity volumetric coverage (e.g., as represented in athree-dimensional CAD model) is produced on the substrate 104. In somearrangements described below, LENS™ can be used for surface treatmentsof metals in which ceramic powder can be fed into a laser-generatedmolten metal pool to form a metal-ceramic composite. In one embodiment,a 0.5 kW continuous wave Nd:YAG laser beam can be used to coat TCPparticles on cp-Ti.

Calcium Phosphate Coating Example 6

In this example, commercial grade calcium phosphate powder with HAp as aprimary phase (Monsanto, Calif.) having a particle size ranging from 45to 150 μm was used to coat a 0.89 mm thick Ti substrate (Alfa Asear) of99.7% purity. Average specific surface area of the precursor powder wasdetermined by the Brunauer, Emmett and Teller (BET) method (5 pointanalyzer, Tristar Micromeritics, USA) after degassing at 350° C. with acontinuous flow of nitrogen. The Ti substrate was cleaned with acetoneto remove organic materials from the surface prior to coating. A LENS750 (Optomec, Albuquerque, N. Mex., USA) unit with a 0.5 kW continuouswave Nd:YAG laser was used to coat Ti substrates. During laserfabrication, the top surface of the Ti metal substrate was melted andTCP powder was fed to the molten metal region with the help of a carriergas (e.g., Argon). The molten metal, along with the trapped TCP powder,solidified rapidly as the laser head moves across the substrate. Toreduce the oxidation of Ti, the coatings were fabricated in a controlledatmosphere with total O₂ content less than 10 parts per million (ppm) inan atmospheric chamber.

Laser power, scan speed and powder feed rate can be varied duringsynthesis of the coatings. Specific examples of LENS™ parameters thatcan be used are listed in Table 3. In some examples described below, Tisubstrates were coated with TCP using 400 W or 500 W laser power (energydensity of the laser beam can be 224 W/mm² and 280 W/mm², respectively)and with a scan speed of 10 mm/sec. or 15 mm/sec. In some examples,powder feed rates of 9 g/min. or 13 g/min. were used. One of ordinaryskill in the art will recognize that the parameters presented in Table 3are exemplary, and that different parameter settings can be selected,e.g., such as settings based on prior optimization for processingdesirable coating characteristics. In one embodiment, single layer TCPcoatings can be synthesized; however, the scanning/coating process maybe repeated many times according to the design requirement.

TABLE 3 Processing parameters used to fabricate TCP coatings LENS ™Parameters Laser Coating Laser Power Scan Speed Powder Feed Rate SingleLayer 400 W or 500 W 10 nm/sec. or 9 g/min. or 13 g/min. 15 nm/sec.

Characterization of TCP Coatings on Ti Substrates Example 7

To analyze the coated Ti products of Example 5, the coated samples werecross-sectioned, mounted and prepared for metallographic observation. Inthis example, top surfaces of the coatings were polished to observe thedistribution of TCP in the coating. The polished sections were etchedwith a solution of hydrofluoric acid (49% by acidometry), nitric acid(15.8 N) and distilled water in a ratio of 1:2:25 to reveal coatingmicrostructure. Microstructural characterization of the top andcross-sectioned surfaces of the coating was performed using a scanningelectron microscope (Hitachi s-570 SEM). Siemens D500 KrystalloflexX-ray diffractometer using copper Kα radiation at 30 kV was used todetermine different phases in the coating. Energy dispersivespectroscopy (EDS) was also used for qualitative chemical microanalysisof the coating surface. Further, Vicker's microhardness (Leco, M-400G3)measurements were carried out on transverse sections of the coating byapplying a 200 gm load for 10 seconds, and an average of 5 measurementson each sample are reported.

Coating Thickness

In the examples described below, the thickness of the TCP coating wasfound to be influenced by laser power, scan speed and powder feed rate.

Example 8

FIGS. 6A-B are SEM micrographs of TCP coating layers on Ti substratesfabricated using LENS™ at a scan speed of 15 mm/sec. with a powder feedrate of 13 g/min. and at (A) 500 W laser power, and (B) 400 W laserpower in accordance with an embodiment of the disclosure. Thecross-sectional micrographs of TCP coatings shown in FIGS. 6A-B indicatevariations of coating thickness with varying laser power. For example,the coating thickness increased from 250 μm to 400 μm when the laserpower was increased from 400 W to 500 W.

Example 9

In this example, the thickness of the TCP coating was found to beinfluenced by laser scan speed. FIGS. 7A-B are SEM micrographs of TCPcoating layers on Ti substrates fabricated using LENS™ at 500 W powerwith a powder feed rate of 14 g/min. and a scan speed of (A) 10 mm/sec.,and (B) 15 mm/sec. in accordance with an embodiment of the disclosure.Increasing the scan speed from 10 mm/sec. to 15 mm/sec. while keepingthe laser power at a constant dc voltage of 500 W resulted in a decreaseof the coating thickness by a range of about 60-80 μm. However, thecoating was uniform with respect to TCP distribution and concentrationover the entire region.

Example 10

In this example, the thickness of the TCP coating was also found to beinfluenced by TCP powder feed rate. FIGS. 8A-B are SEM micrographs ofTCP coating layers on Ti substrates fabricated using LENS™ at 500 Wpower at a scan speed of 10 mm/sec. and with powder feed rate of (A) 9g/min., and (B) 13 g/min. in accordance with an embodiment of thedisclosure. As shown in FIGS. 8A-B, the thickness of the coatingdecreased by approximately 100 μm when the powder feed rate wasdecreased from 13 g/min. to 9 g/min.

Microstructure and Volume Fraction of TCP Example 11

In one embodiment, the loading of TCP particles in the coating can alsobe significantly affected by a combination of laser scan speed andpowder feed rate. FIGS. 9A-B are interfacial SEM micrographs of TCPcoating layers on Ti substrates fabricated using LENS™ at 500 W powerwith a powder feed rate of 13 g/min. and a scan speed of (A) 10 mm/sec.,and (B) 15 mm/sec. in accordance with an embodiment of the disclosure.At a slower scan speed (shown in FIG. 9A), a higher volume fraction ofTCP was observed in the coatings compared to coatings made at a higherscan speed (see FIG. 9B).

Example 12

FIGS. 10A-B are SEM micrographs of cross-sections of the microstructureof TCP coating layers on modified Ti substrates fabricated using LENS™at 500 W power, 10 mm/sec. scan speed and 9 g/min. powder feed rate andshowing a composite coating layer region (A) close to the metalsubstrate, and (B) along an exterior surface of the coating layer inaccordance with an embodiment of the disclosure. Referring to FIGS.10A-10B, within the metal-bioceramic composite layer, the microstructurechanges from columnar Ti grains at the lower region of the compositelayer to equiaxed grains near the exterior surface of the compositelayer. The lower region of the coating layer (e.g., closest to the metalsubstrate) shows columnar grain growth of Ti with TCP particles alongthe grain boundaries (shown in FIG. 10A). Moreover, the interfacebetween the composite coating layer and the metal substrate appearsdiffused. FIG. 10B shows the equiaxed grains at and near the exteriorsurface of the composite coating layer. In this region the TCP particleswere found to align along the grain boundaries. Moreover, the volumefraction of TCP in this region was greater when compared to the lowerregion nearest the metal substrate. The variation of grain structure isprimarily due to the concentration of TCP during solidification of Ti.

Example 13

FIG. 11 is a SEM micrograph of a Ti surface modified with a TCP coatinglayer using LENS™ at 400 W laser power, 15 mm/sec. scan speed and 13g/min. powder feed rate, and after polishing and etching in accordancewith an embodiment of the disclosure. As shown in FIG. 11, the compositecoating surface is crack-free and contains uniformly distributed TCPparticles. Moreover, the coating surface has a rough appearance, whichin some embodiments, can be advantageous to bone tissue bonding in vivo.In this example, polishing and etching reveals the distribution of TCPin the coating layer. It can be seen from FIG. 11 that TCP is evenlydistributed in the coating with sizes ranging from 8-12 μm. Usingcontrast imaging software (not shown), it was found that almost 65-70%of the surface consists of TCP. Therefore, a gradient in TCP volumefraction from 0% to at least 65% by volume was achieved within 250 μmcoat thickness. Additionally, this example illustrates that TCP canextend across the surface of the composite coating layer and can have acontinuous structure.

Substrate evolution from a liquid metal to a microstructural compositelayer is governed by the ratio of a temperature gradient (G) to asolidification rate (R), i.e.; (G/R) [2]. The growth rate of amicrostructure in a composite layer can be determined by the amount ofundercooling below the melting point of the specific metal (e.g., ametal casting phenomenon). Due to rapid heat transfer at the interfaceof molten Ti and unmelted Ti, the rate of nucleation of the metalpredominates over the rate of growth of the microstructure. This effectcan result in a thin region of fine Ti grains located at themetal-coating layer interface. As the effect of undercooling diminishesas the distance away from the unmelted Ti interface increases, the rateof nucleation of the molten metal decreases. For example, as the latentheat of fusion is being released, the more exterior positioned liquid Tican remain in a molten state. Moreover, TCP particle buoyancy canfurther reduce the TCP load near the interface as well as furtherpromote microstructure growth at increasing distances away from theinterface. This microstructure growth is controlled by the rate of heattransfer from the interface. Because the above-described phenomenonestablishes a temperature gradient towards the interface, themicrostructure growth occurs in a direction opposite to the heat flow.The effect is the formation of columnar grains at the interface [4].These columnar grains advance towards the surface of the coating.However, near the surface of the composite coating layer, heterogeneousnucleation is a predominating factor due to the presence of a highvolume fraction of TCP. As a result, the exterior upper region of thecomposite coating layer consists of mostly fine equiaxed grainstructures.

As mentioned, the density difference between TCP and Ti can play a rolein the distribution of TCP. Because the density of TCP (3.07 g/cc) islower than the density of Ti (4.5 g/cc), the TCP particles may exhibitbuoyancy behavior in the molten Ti metal pool. The movement of thesolid-liquid interface during the solidification process, which isgoverned by the cooling rate, can determine the entrapment of TCP in thevarious sub-layers of the composite coating [5, 6]. At the lower regionsof the composite coating layer (e.g., near or at the coating-substrateinterface), where the solidification rate is high, the fast advancementof the solid-liquid interface can push the TCP particles upward leadingto a lower volume fraction of TCP near the metal substrate (e.g., at ornear the coating-substrate interface. While ascending through the moltenmetal pool, the solidification rate and the movement of the solid-liquidinterfaces decreases. The slower movement of this interface can entrap agreater amount of TCP near and at the upper, exterior regions of thecomposite coating.

Hardness of the Coating Layers Example 14

FIG. 12 is a graphical representation of hardness profiles of TCPcoating layers formed using LENS™ at 500 W laser power and a powder feedrate of 13 g/min. in accordance with an embodiment of the disclosure. Asshown, the microhardness of the TCP coatings increase from 200±18 Hv atthe coating-metal substrate interface to an average value of 902±200 Hvin the upper, exterior regions of the composite coating. In thisexample, the gradual increase in hardness from the coating-metalsubstrate interface to the upper, exterior regions of the coating layercan be a result of micro-structural variation across the coatingthickness.

The combined effect of fine equiaxed grains and a high volume fractionof TCP at the exterior regions of coating layer can result insignificantly greater hardness values. Hardness of the coating layer inthe intermediate regions may be attributed to the formation of aparticulate composite of TCP and Ti. According to the microstructuralanalysis described above with respect to Examples 11-13, the decrease inhardness at or near the coating-metal substrate interface can beattributed to elongated, columnar Ti grains and a low volume fraction ofTCP.

The physical properties (e.g., thickness, volume fraction of TCP) andmicrohardness data for the TCP coatings, prepared with different laserparameters, are summarized in Table 4.

TABLE 4 Compilation of coating layer properties as a function of LENS ™parameters LENS ™ Parameters Ave. Laser Laser Scan TCP Coating PowerSpeed Powder Feed Thickness Volume Hardness (W) (mm/sec.) Rate (g/min.)(μm) Fraction (Hv) 400 15 13 250 Low 706 ± 25  500 15 13 400 High 781 ±70  500 10 13 375 High 902 ± 180 500 10 9 220 Low 648 ± 118

Composition and Phase Analysis

In one embodiment, the presence of interconnected pores within the TCPpowder provides high powder surface area. For example, the TCP powderhaving a particle size ranging from 45 to 150 μm, showed a BET surfacearea of 58.2±4.6 m²/g.

Example 15

FIG. 13 is a graphical representation of X-ray diffraction spectra ofTCP coating layers using LENS™ and with a powder feed rate of 13 g/min.in accordance with an embodiment of the disclosure. As shown in FIG. 13,the dominant characteristic peaks were identified as Ti and TCP. Duringlaser interaction, TCP is generated by the high temperature processingof precursor calcium phosphate as hydroxyapatite primary phase. A smallpeak corresponding to calcium titanate (CaTiO₃) was also observed in thecoating. It was found that the formation of this calcium titanate phasewas independent of laser parameters. The sharp peaks in the XRD patternsindicate that the TCP in the coatings are mostly crystalline in nature.Additionally, the presence of TCP particles on the surface was confirmedby EDS analysis (not shown).

Dissolution kinetics of a material is governed by its crystallinity withother solution parameters like temperature, pH, etc. Calciumphosphate-based ceramic shows higher dissolution tendency with adecrease in crystallinity. It is desirable to have a highly crystallineTCP phase in the coating because the crystalline structure can providethe coating with more stability. In contrast to Ti, TCP is a non-laserabsorbing material and does not melt during laser processing usingLENS™. The LENS™ process is very different from traditional reactionheating methods due to its unique characteristics such as an ultrahighheating rate and a rapid solidification rate. Due to thesecharacteristics, TCP retains its crystallinity in the coating, which isa property difficult to achieve in commercial coating processes likeplasma spray.

As previously explained, during the LENS™ process, the laser beam isfocused onto the surface of the Ti substrate. The high energy of thelaser beam locally melts the Ti surface and creates a molten pool whichis maintained during the laser irradiation. Simultaneously, the addedpowder material injected into the molten pool partially melts byinteraction with the laser and also the hot molten metal. Due to thehigh temperature processing, HAp phase in the added powders reacts toform TCP. The high temperature process also induces interactions betweenHAp and TiO₂ leading to formation of various compounds. High temperaturephases that can form between CaO and TiO₂ are Ca₃Ti₂O₇, CaTiO₃,Ca₄Ti₃O₁₀ and Ca₅Ti₄O_(3 [)2, 3]. For example, possible reactionsbetween HAp and Ti are as follows:

HAp+TiO₂→CaTiO₃+TCP+H₂O

TCP+TiO₂→CaTiO₃+α-Ca₂P₂O₇

α-Ca₂P₂O₇+Ti→CaTiO₃+CaO+P₂O₃(g)↑

The presence of CaTiO₃ was detected in the XRD pattern (shown in FIG.13). However, the amount of this phase formation can be governed by theinteraction time between Ti and TCP. Since LENS™ is characterized byrapid heating and cooling rates, the interaction time is relativelyshort. Therefore, by optimally choosing appropriate LENS™ parameters,these coatings can be made with a highly crystalline TCP phase alongwith a small amount of other phases.

Morphology of OPC1 Cells on TCP Coating

In one embodiment, evaluation of the biological activity of TCP coatingscan be performed using TCP-coated Ti substrate samples seeded with humanosteoblast cells (HOCs).

Example 16

In this example, the Ti samples were sterilized by autoclaving at 121°C. for 20 minutes. The cells used in this example were derived from animmortalized, osteoblastic precursor cell line (OPC1) established fromhuman fetal bone tissue. For routine maintenance, cells were plated at adensity of 10⁵/cm² in 100 mm tissue culture plates (Cellstar, GreinerBio-one, Germany) and cultured in McCoy's 5A Medium (enriched with 5%fetal bovine serum, 5% bovine calf serum and supplemented with 4 μg/mlof fungizone). The cells were seeded onto coated Ti samples and uncoatedTi control samples placed in 6 well plates and maintained at 37° C.under an atmosphere of 5% CO₂ and 95% air. The culture medium in theplates was changed every 2 days. In this example, all OPC1 cellsoriginated from the same cell line passage and all plates were kept inidentical conditions.

The coated and uncoated Ti samples were removed from culture aftereither 5 or 11 days of cell-sample co-incubation. Morphology of the OPC1cells were subsequently assessed by SEM. Samples were rinsed with 0.1 Mphosphate-buffered saline (PBS) and subsequently fixed with 2%paraformaldehyde/2% glutaraldehyde in 0.1 M cacodylate buffer overnightat 4° C. Following a rinse in 0.1 M cacodylate buffer, each sample waspostfixed in 2% 6 osmium tetroxide (OsO₄) for 2 hours at roomtemperature. Fixed samples were then dehydrated in an ethanol series(e.g., 30%, 50%, 70%, 95% and 100%) three times, followed by ahexamethyl-disililane (HMDS) drying procedure. Dried samples were goldcoated (Technics Hummer, San Jose, Calif.), and observed using a Hitachis-570 SEM.

FIGS. 14A-D are SEM micrographs illustrating OPC1 morphology and celladhesion on Ti substrates after 5 days incubation in cell culture with(A) uncoated Ti, and (B) TCP coated Ti; and after 11 days incubation incell culture with (C) uncoated Ti, and (D) TCP coated Ti in accordancewith an embodiment of the disclosure. When comparing OPC1 morphology andcell adhesion following 5 days of incubation, there are more OPC1 cellsattached to the surface of the TCP coated substrates than the uncoatedsubstrates. For example, cells attached to the TCP coating, as shown inFIG. 14B, have cell edges with a diffuse, spread-like morphology withseveral lamellipodia and filopodia extensions, while FIG. 14A shows thatthe uncoated Ti surface has very little cell spreading. This cellattachment trend continues with additional incubation time. For example,cell layers were formed on TCP coatings after 11 days of culture, asshown in FIG. 14D. Additionally, extracellular matrix (ECM) (seen asspherical granules on the surface of the cells, which is an indicationof mineralization) was formed by the cells attached to the TCP-coated Tisubstrates, but not by the cells attached to the uncoated Ti samples.

Cell Survival and Proliferation

Cell survival and proliferation can also be considered in adetermination of metal implant material in vitro biocompatibility.

Example 17

In this example, cell proliferation of OPC1 cells on coated and uncoatedTi samples were assessed by MTT assay (Sigma, St. Louis, Mo.). Similarlyto the MTT assay described above with respect to Example 4, an MTTsolution of 5 mg/ml was prepared by dissolving MTT in PBS followed byfilter sterilization. The MTT solution was diluted (50 μl into 450 μl)in serum free, phenol red free Dulbeco's Minimum Essential medium (DME).Diluted MTT solution was then added to each sample. Following 2 hours ofsample incubation with OPC1 cells in the MTT solution, a solventsolution made up of 10% Triton X-100, 0.1 N HCl and isopropanol wasadded to dissolve the resultant formazan crystals. In this example, 100μl of the resulting supernatant was transferred into a 96-well plate andread by a plate reader at 570 nm. Data are presented as mean±standarddeviation.

FIG. 15 is a graphical representation of OPC1 cell proliferation on TCPcoated Ti substrates and uncoated Ti substrates in accordance with theabove-described MTT assay. As shown in FIG. 15, cell proliferation wasevident over the duration of the experiment (e.g., over 3, 5 and 8 daysof cell incubation). In the present example, data from the MTT assayshowed that the number of cells on TCP-coated Ti substrates isconsistently greater than the number of cells on uncoated Ti substrates.Statistical analysis was performed using Student's t test, and p<0.05was considered statistically significant for this example. Thestatistical analysis showed significant differences in cellproliferation on the TCP-coated samples after 3, 5 and 8 days in cellculture. The results also demonstrated significant differences in cellproliferation on TCP-coated Ti samples when compared to uncoated Ticontrol samples for all time periods tested.

The physical and biological properties of bioceramic-coated metalsubstrates examined in Examples 6-17 indicate that different coatingproperties can be achieved by suitably selecting the process parameters.For example, a bioceramic composite coating prepared using a high laserpower, high powder feed rate and low scan speed can yield a high volumefraction of coating material having a high level of microhardness andgood bone cell-materials interactions.

Preparation of Calcium Phosphate Coating with Deposited Ag Example 18

In this example, commercial grade calcium phosphate powder, mainly HApas primary phase, having particle size ranging from 45 to 150 μm wasused to coat 0.89 mm thick Ti substrate (Alfa Asear) of 99.7% purity. Tisubstrate was first cleaned with acetone to remove organic materialsfrom the surface prior to coating. LENS™ 750 (Optomec, Albuquerque, N.Mex., USA) unit with 0.5 kW continuous wave Nd:YAG laser was used toprocess TCP coatings on Ti substrate. Detailed discussion of TCP coatingon Ti using LENS™ has been discussed earlier [15]. TCP coated sampleswere cleaned with acetone and distilled water prior to Agelectrodeposition. The electrodeposition was performed from an aqueoussolution of AgNO₃ at 5 V for 2 min. using platinum as anode. Differentconcentrations of silver nitrate solution were used as electrolyte forthe Ag-electrodeposition and are summarized in Table 5.

TABLE 5 Deposition conditions of Ag deposited samples Serial numberDeposition condition S1 0.001M AgNO₃ solution S2 0.1M AgNO₃ solution S30.5M AgNO₃ solution S4 No Ag coating

As the deposition voltage and time remains constant, differentconcentrations of AgNO₃ solution will deposit a varying amount of Ag onthe TCP coated surfaces. Surface and cross-sectional morphology of thecoating was studied using scanning electron microscope (SEM) fitted withan energy dispersive spectroscopy (EDS) detector (not shown).

Antimicrobial Tests Example 19

To determine antimicrobial activity, TCP coated samples, with andwithout deposited silver, from Example 18 were challenged individuallywith Pseudomonas aeruginosa and Staphylococcus aureus. Samples wereplaced in a multi-well plate (FALCON brand), with one sample per welland appropriately labeled. Trypticase soy agar (TSA) culture medium wasused to prepare TSA plates for growing surviving bacterial colonies. Forbacterial dilutions, sterile saline (0.85% sodium chloride in water) wasused. The media M101, consisting of M110 plus 0.1% yeast extract, 10%glucose, 0.1% neopeptone, 25% M101 and 1.0% Bovine serum in water wasused as challenge media.

To each well 4 ml of inoculum prepared in M101 or M110 medium (for P.Aeruginosa and S. aureus, respectively) was added. The inoculum wasprepared by adding overnight culture of bacteria to M101 or M110 mediumin proper dilution that would yield an initial inoculum of ˜1×10⁵cfu/ml. The multi-well plate was then incubated at 37° C. for 24 h. Thefluid from each well was appropriately diluted and plated on TSA plates.Coated samples were plated at 10⁻¹, 10⁻² and 10⁻³ dilutions. The zerotime point samples and control samples (e.g., S4 samples from Table 5)were plated at 10⁻³, 10⁻⁵ and 10⁻⁷ dilutions. The samples were incubatedat 37° C. for 48 h and surviving colonies counted. From the initialinoculum dose obtained from zero time plate count and surviving bacteriacount, log reduction values were calculated.

Table 6 shows the antimicrobial efficacy of the TCP and Ag-TCP coatingsfollowing a 24 hour Pseudomonas aeruginosa and Staphylococcus aureuschallenge assays. The TCP coatings having no Ag deposition (S4), show noantimicrobial activity against P. aeruginosa or S. aureus. However, theAg-TCP coatings S2-S3 show strong antimicrobial activity against bothbacterial strains, and sample S1 shows strong antimicrobial activityagainst P. aeruginosa. Log reduction in bacteria due to Ag coating isestimated as logarithm of ratio of initial bacterial colonies to averagefinal surviving colonies. Log reductions>4, equates to a>99.99%reduction in bacteria, is counted as strong antimicrobial activity.Therefore, all of the Ag-TCP coatings show very strong antimicrobialactivity towards Pseudomonas aeruginosa, whereas TCP coatings without Agshow an increase in bacterial colonies. A reduction in antimicrobialactivity can be noticed for samples S1 with low Ag content when comparedto samples (S2, S3) with greater Ag content. However, as shown in Table6, the antimicrobial activity does not improve with Ag depositionsolutions having greater than 0.1 M AgNO₃ solution, as evidenced by thesimilarity in results between S2 and S3 samples.

TABLE 6 Antimicrobial efficacy of the silver treated titanium discs. A24 h Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC33591 challenge assay. Values are the log reduction obtained as anaverage of a triplicate assay. Log of zero time inocula Log survivors(triplicate) Log reduction P. aeruginosa S. aureus P. aeruginosa S.aureus P. aeruginosa S. aureus Sample ATCC 9027 ATCC 33591 ATCC 9027ATCC 33591 ATCC 9027 ATCC 33591 S1 5.5 5.8 1.78 5.3 5.41 2.48 S2 5.5 5.81.48 2.66 5.71 5.12 S3 5.5 5.8 1.48 2.78 5.71 5.00 S4 5.5 5.8 7.18 7.78N/A N/A

Although Ag is well known as a strong antimicrobial agent, highconcentrations of Ag have been reported to be cytotoxic. It is reportedthat the toxicity of Ag ions affect the basic metabolic cellularfunctions of all specialized mammalian cells. Therefore, it is useful toincorporate a minimum amount of Ag on medical implant surfaces tominimize this tissue cytotoxicity effect, while maintaining a high levelof antimicrobial activity. In accordance with an embodiment of thedisclosure, the Ag-TCP coating prepared from 0.1M AgNO₃ solution has anenhanced antimicrobial property combined with good natural bone cellproliferation.

Discussion of LENS™ Processing

Conventional plasma spray processes produce a coating of 150-200 μmthick. In contrast, TCP coatings resulting from LENS™ processing canhave thickness varying from approximately 200-700 μm, or in anotherembodiment, from approximately 100-900 μm. Moreover, the volume fractionof TCP in the coating can be tailored by suitably selecting theprocessing parameters like laser power, laser scan speed and powder feedrate (see Tables 3 and 4). While plasma spray processes can only producea thick TCP layer on top of the metal substrate, LENS™ processing canproduce a compositional gradient coating. By using a LENS™ coatingprocess, deposition of successive layers (e.g., creating a multilayercoating) can be used to develop a compositional gradient coatingstarting from pure Ti at the core to pure TCP on the surface with aTi-TCP intermixed region in between.

As described above, human osteoblast cell behavior on the TCP coatingwas characterized by an OPC1 cell morphology study using SEM. Cellattachment is one stage of bone cell-materials interaction. Cells attachto and migrate across the material surfaces by a variety of cellularmechanisms such as extension of filopodia and lamellipodia. In oneexample, cell morphology, as examined by SEM, showed that filopodiaextensions from the cells to the substrate are more pronounced for TCPcoated surfaces. This result demonstrates that TCP coating promotesbetter cell attachment and spreading behavior compared to uncoated Ti.SEM observation also revealed the presence of extracellular matrix (ECM)on the TCP coatings, which can be an indication of cell differentiation.For example, the formation of ECM indicates that OPC1 cells on TCPcoatings are capable of producing a matrix suitable for mineralizationand can suggest the initiation of an intracellular biomineralizationpathway. Another determining factor of successful bone cell-materialsinteractions can be cell proliferation, which is also an indication ofcell survival. As described above, the MTT assay and SEM observationsdemonstrated that the TCP coating is non-toxic, and thus, does notinhibit cell proliferation.

D. REFERENCES

The following references are herein incorporated by reference.

-   [1] Gefen, A., Med. Biol. Eng. Comput., 2002; 40:311-322.-   [2] Wang Y C, Li Y M, Yu H L, Ding J, Tang X H, Li J G, Zhou Y H.    Surf & Coat Tech., 2005; 200:2080-2084.-   [3] Lusquiños F, Pou J, Arias J L, Boutinguiza M, Leon B, Perez-Amor    M, Driessens F C M, Merry J C, Gibson I, Best S, Bonfield W. J Appl    Phys., 2001; 90 (8):4231-4236.-   [4] Heine R W, Loper C R, Rosenthal P C. McGraw-Hill Book Company    1967.-   [5] Chao M J, Niu X, Yuan B, Liang E J, Wang D S. Surf & Coat Tech.,    2006; 201:1102-1108.-   [6] Yang S, Chen N, Liu W, Zhong M, Wang Z, Kokawa H. Surf & Coat    Tech., 2004; 183:254-260.-   [7] Gong D, Grimes C A, Varghese O K, Chen Z, Hu W, Dickey E C, J.    Mater. Res., 2001; 16:3331-3334.-   [8] V. Parkhutik P, Shershulsky V I, J. Phys. D: Appl. Phys., 1992;    25:1258-1263.-   [9] Das K, Ph.D Thesis, Washington State University, May 2007.-   [10] Zhang Z W, Rare Metal Mater. Eng. 1 1996; 1:49.-   [11] Peterwig H G, Pharmacol. Ther., 1996; 69:127.-   [12] Rahn R O, Landry L C, Photochem. Photobiol., 1973; 8:29.-   [13] Valle B O, Ulmer D D, Annu Rev. Biochem., 1972; 41:91-128.-   [14] Ritchie J A, Jones C L, Lett. Appl. Microbiol., 1990; 11:152.-   [15] Roy M, Krishna B V, Bandyopadhyay A, Bose S, Acta    Biomaterialia, 2008; 4 (2): 324-333.-   [16] Das K, Bose S, Bandyopadhyay A, Acta Biomaterialia, 2007; 3    (4): 573-585.

E. CONCLUSION

The present disclosure describes methods for modifying the surfaces ofmetal implantable materials, such as titanium (Ti) substrate surfaces,and the modified metal materials formed therefrom. Some of theembodiments of modified metal implantable materials were evaluated fortheir respective physical and biological properties, including thoseproperties useful for surgical implants. In one embodiment, metalimplantable materials (e.g., Ti metal materials) can be oxidized (e.g.,thermally oxidized, electrochemically oxidized) to form a titanium oxidelayer on the material surface. For example, the metal implantablematerial can be anodized in an electrolytic solution containing sodiumfluoride and sulfuric acid, to form a titania nanotube morphology on thesubstrate surfaces. The oxidized surface, with or without silverdeposition, can provide an improved surface for cellular attachment,supported high cellular proliferation rates and enhanced bonecell-materials interactions in comparison to the cellular behaviorassociated with non-modified Ti-control surfaces. Furthermore, silver(Ag) coated surfaces demonstrate antimicrobial properties as evidencedby their ability to effectively inhibit greater than 99% of Pseudomonasaeruginosa colony growth. In contrast, Ti substrate surfaces, with orwithout TiO₂ nanotube microstructures or films, demonstrated noinhibitory properties against colony formation and growth of P.aeruginosa.

In other embodiments, methods disclosed herein can be used for formingcalcium phosphate-based bioceramic coatings on metallic implants. In oneembodiment, a LENS™ process can be used to apply a calcium-basedbioceramic composite coating layer to metal surfaces. In someembodiments, a coating thickness of approximately 50 μm to 900 μm havinga desirable and/or selectable amount of calcium-based bioceramicmaterial distributed within the coating can be achieved. Additionally,the LENS™ process can be used to create coatings having a compositiongradient across the coating thickness. Such composition gradients cansignificantly reduce the interfacial problems associated with a sharpinterface, such as those present when using conventional coatingprocesses (e.g., plasma spray). Microstructure formation varies withinthe composite coating layer created using the LENS™ process (e.g.,columnar Ti grain structures can be more prevalent in regions closer tothe composite coating-metal interface and equiaxed Ti grain structurescan be more prevalent in regions closer to the exterior of the coatinglayer).

Microstructure variance along a coating layer thickness can be, at leastin part, due to the ratio of temperature gradient to solidification rate(G/R) generated during the LENS™ process. In some embodiments, themicrostructure variance and bioceramic loading can increase a coatinglayer hardness value. In one specific example, formation of fine Tigrain structures, along with TCP presence at the grain boundaries,increased the coating hardness to approximately 1049±112 Hv compared toan unmodified substrate hardness of 200±15 Hv. In vitro studies indicatethat calcium-based bioceramic composite coatings, such as TCP coatings,have good biocompatibility with OPC1 cells. For example, the coatingsurface promotes OPC1 cell attachment and proliferation. In somearrangements, the coating can also promote biomineralization.

In accordance with some arrangements, a metal implant and/or metalsubstrate can include a calcium-based bioceramic coating with varyingthickness. For example, the coating may have a first thickness at afirst surface region of the metal substrate and a second thickness at asecond region of the metal substrate. Moreover, the coatingcharacteristics, other than thickness, at a first region of the metalsubstrate (e.g., microhardness, microstructure, volume fraction ofbioceramic particles, and other biophysical and biochemical properties)can be the same or different from other regions of the metal substrate.

Furthermore, a bioceramic coating on the surface of a metal implant canhave a complex pattern of modified regions and unmodified regions aswell as have differences in the resulting modifications between theregions. In one embodiment, one or more regions of a metal implant maybe modified with a titania nanotube microstructure with a silvercoating, and in another region of the metal implant, a bioceramiccoating can be formed. In other embodiments, regions modified with atitania nanotube microstructure with a silver coating can be partiallyor completely overlapping with regions having a bioceramic coating.

Various embodiments of the technology are described above. It will beappreciated that details set forth above are provided to describe theembodiments in a manner sufficient to enable a person skilled in therelevant art to make and use the disclosed embodiments. Several of thedetails and advantages, however, may not be necessary to practice someembodiments. Additionally, some well-known structures or functions maynot be shown or described in detail, so as to avoid unnecessarilyobscuring the relevant description of the various embodiments. Althoughsome embodiments may be within the scope of the claims, they may not bedescribed in detail with respect to the Figures. Furthermore, features,structures, or characteristics of various embodiments may be combined inany suitable manner. Moreover, one skilled in the art will recognizethat there are a number of other technologies that could be used toperform functions similar to those described above and so the claimsshould not be limited to the devices or methods described herein. Whilesome processes are described in a given order, alternative embodimentsmay perform methods having steps in a different order, and someprocesses may be deleted, moved, added, subdivided, combined, and/ormodified. Accordingly, each of these methods may be implemented in avariety of different ways. Also, while some methods (e.g., surfacemodification methods) are at times shown as being performed in series,these methods may instead be performed in parallel, or may be performedat different times. The headings provided herein are for convenienceonly and do not interpret the scope or meaning of the claims.

The terminology used in the description is intended to be interpreted inits broadest reasonable manner, even though it is being used inconjunction with a detailed description of identified embodiments.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words “comprise,” “comprising,” and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in a sense of “including,but not limited to.” Words using the singular or plural number alsoinclude the plural or singular number, respectively. When the claims usethe word “or” in reference to a list of two or more items, that wordcovers all of the following interpretations of the word: any of theitems in the list, all of the items in the list, and any combination ofthe items in the list.

Any patents, applications and other references, including any that maybe listed in accompanying filing papers, are incorporated herein byreference. Aspects of the described technology can be modified, ifnecessary, to employ the systems, functions, and concepts of the variousreferences described above to provide yet further embodiments.

These and other changes can be made in light of the above DetailedDescription. While the above description details certain embodiments anddescribes the best mode contemplated, no matter how detailed, variouschanges can be made. Implementation details may vary considerably, whilestill being encompassed by the technology disclosed herein. As notedabove, particular terminology used when describing certain features oraspects of the technology should not be taken to imply that theterminology is being redefined herein to be restricted to any specificcharacteristics, features, or aspects of the technology with which thatterminology is associated. In general, the terms used in the followingclaims should not be construed to limit the claims to the specificembodiments disclosed in the specification, unless the above DetailedDescription section explicitly defines such terms. Accordingly, theactual scope of the claims encompasses not only the disclosedembodiments, but also all equivalents.

We claim:
 1. A method for modifying a surface of a metal device forimplanting in a body, the method comprising: oxidizing a first metalsurface of the device to form a titanium oxide layer comprising titaniumoxide nanotubes with an inner diameter greater than 100 nm on at least aportion of the first metal surface to enhance bone cell interactionswith the metal device; and depositing silver onto the first metalsurface of the device for inhibiting microbial growth adjacent to theimplantable metal device.
 2. The method of claim 1, further comprising:melting a second metal surface with a laser to form a molten secondmetal surface; feeding a calcium phosphate powder at a first feed rateonto the molten second metal surface; and solidifying the molten secondmetal surface including the powder to form a calcium phosphate-basedbioceramic surface coating.
 3. A method for modifying a surface of ametal device for implanting in a body, the method comprising; meltingthe surface of the metal device using a Laser Engineered Net Shaping(LENS™) processing laser to form a molten metal pool at the surface; anddepositing calcium-based bioceramic powder onto the molten metal pool,wherein the powder mixes with the molten metal pool and solidifies toform a surface coating for enhancing bone cell interactions with themetal device.
 4. The method of claim 3 wherein the metal device includestitanium metal, and wherein the surface coating includes a titanium andcalcium phosphate-based bioceramic gradient extending from acoating—metal interface to an exterior region of the surface coating. 5.The method of claim 3 wherein the surface coating has a coat thicknessof approximately 100 μm to approximately 900 μm.
 6. The method of claim3 wherein following the depositing calcium-based bioceramic powder ontothe molten metal pool, the method further comprises depositing anantimicrobial agent onto at least an exterior surface of the surfacecoating for inhibiting microbial growth adjacent to the implantablemetal device.